The cleavage cycles of early metazoan embryos are limited to the bare essence of genome replication and segregation, lacking the growth, transcription and checkpoints which embellish the somatic cell cycle. These cleavage cycles are therefore the natural framework upon which to construct models of the much more complicated somatic cell cycles. Such models are the intellectual foundation for thousands of laboratories world-wide intent on understanding cell division and growth, and how to prevent, counteract and treat their misregulation. But the true nature of the most minimal cell cycle, the metazoan cleavage cycle, is far from fully understood. In particular, the regulatory modules that are widely thought to initiate mitosis are not important in the cleavage cycles. The unknown mechanism that truly controls entry into mitosis in these minimal cleavage cycles, and which by extension could be extant in all metazoan cell cycles, remains obscure. We propose that this unknown mechanism, and perhaps many other aspects of the minimal cell cycle, could be revealed by comprehensive analyses. The molecular landscape of the cleavage cycles can only be generated by MS- based proteomics, particularly because the virtual absence of zygotic transcription makes these cleavage cell cycles refractory to transcriptomics. In striving towards radically improved models of the cell cycle, we aim to define the minimal animal cell cycle by i) detecting which proteins and which phosphorylation sites oscillate in a cell cycle dependent manner, ii) quantifying the extent of variations/oscillations, iii) provide information about absolute phosphorylation stoichiometries, and iv) provide absolute quantities for key cell cycle regulators and their post-translational modifications (not limited to phosphorylation) that mediate the minimal cell cycle. This study will be the first large-scale, quantitative proteomic study of cleavage cycles and the first global analysis of the metazoan cell cycle that doesn't rely on drug-based synchronization techniques. The embryos of the frog X. laevis embryos provide a compelling context for these experiments due to their large size, holoblastic cleavage, and the capability for naturally synchronized cell cycles. The proposed experiments have the potential for revolutionary insights into the cell cycle, as well as generating a trove of data which can be mined and further extended upon by the vibrant cell cycle community. PUBLIC HEALTH RELEVANCE: Instead of using artificial in vitro cultured systems, it is our aim to study the in vivo minimal embryonic cell cycle in order to decipher the true nature of its regulation. Because we aim to describe hundreds of changes in protein abundance and protein modifications that underlie this cycle in vivo with an unprecedented temporal resolution, this project will challenge, refine, and enlarge current models of the cell cycle that are central to our fundamental understanding of proliferation, and to many human developmental diseases and cancers. Apart from furthering our fundamental understanding of the cell cycle, the development of preventatives, treatments and therapies for/of these diseases and malignancies will benefit greatly from this study.